Many soldiers return home with contusive spinal cord injuries (SCI), mostly due to the powerful explosives used in the improvised bombs that rattle U.S. troops inside heavily armored vehicles. In fact, One in Six Wounded in Afghanistan War Have Spinal Cord Injuries (The Spinal Post, Nov.11, 2009). Currently, there is no cure and consequently studies using animal models of SCI are imperative. Results of recent studies, including our own, revealed that there are at least three major factors known to limit functional recovery from incomplete SCI in adults: (1) the presence of axonal growth/regeneration inhibitors associated with the glial scar, (2) the lack of neurotrophin support, and (3) the decreased excitability and plasticity in pre- existing pathways to neurons in damaged spinal cord. In attempts to improve synaptic connectivity in the damaged spinal cord and examine if this will facilitate recovery of motor function after SCI, we have recently utilized a model of unilateral hemisection (HX) of the spinal cord in adult rats. This is convenient experimental model because one side of the cord is lesioned and other remains intact. Using this HX model we have recently developed (through studies funded by our initial Merit Award; 2008-2012), a novel additive treatment designed to neutralize axonal growth/ regeneration inhibitors, deliver neurotrophins and enhance plasticity in damaged spinal cord. We found that this combination treatment (in contrast to treatment with any single component) can re-establish novel synaptic connections around a lesion in chronically hemisected spinal cord. These physiological findings were supported by the observation of increased regeneration and branching of axons near the injury and better functional recovery. Components of this combination treatment are: (i) degradation of scar-associated inhibitory molecules (CSPGs) with the enzyme chondroitinase-ABC (ChABC), (ii) delivery of neurotrophin NT-3 (administered via engineered fibroblasts) and increased function of NMDA receptors in interneurons and motoneurons (using HSV1 viral vector-mediated expression of NMDA receptor NR2D regulatory subunits). The goal of the current proposal is to translate these principles into a clinically amenable application. The proposed research will be a continuation of our previous work, extended to a more realistic contusive SCI model, with refinement of methods for NT-3 delivery and enhanced spinal cord excitability that are FDA- approved or under clinical trials. This novel triple combination approach will combine Ch-ABC treatment with (i) AAV-mediated transgene delivery of NT-3 (AAV-NT3; intraspinal and intrathecal injections; AAV-mediated gene delivery has been found to be safe in multiple clinical trials) and (ii) repetitive electro-magnetic stimulation applied over intact spinal vertebrae (FDA-approved and widely used for treatment of neurological and psychiatric disorders). Due to increasing numbers of individuals with chronic SCI, we will utilize both acute and chronic mid-thoracic contusion SCI models in adult rats. To evaluate the efficacy of these treatments, we will collaborate with leading scientists in the field and conduct a multidisciplinary examination, including in vivo physiology, anatomy, immunochemistry, electron microscopy and behavior. Using these methods we will study the additive or synergistic effects of the triple therapeutic treatment on (1) strengthening synaptic connections through the injury epicenter to lumbar motoneurons, and then to hindlimb muscles (electrophysiology); (2) anatomical plasticity of fibers accounting for the persistence of the synaptic response after exposure to this novel treatment (anatomical tracing and confocal microscopy); (3) cellular and molecular mechanisms underlying the beneficial effects of the combination treatment (electron microscopy and immunochemisctry to study axon remyelination and distribution of ion channels); and (4) recovery of locomotor function in multiple behavioral tests. Preliminary results of on-going experiments show significant improvements of motor function in rats that have received the novel triple treatment compared to controls after thoracic contusive SCI.